The long term objective of this research is to aid in the development of new chemotherapeutic agents based on the properties of the folate enzyme dihydrofolate reductase and thymidylate synthase. The specific aims for the three year grant period requested are: 1) Characterize the RNA fragments found as constituents of thymidylate synthase purified from methotrexate-resistant Streptococcus faecium. 2) Determine if the RNase sensitive macromolecular component to which thymidylate synthase is attached in crude extracts of methotrexate-resistant S. faecium can be found in methotrexate-sensitive organisms and in Lactobacillus casei strains sensitive and resistant to methotrexate. 3) Determine if other enzymes of deoxynucleotide synthesis such as ribonucleotide reductase and DNA polymerase are associated with the macromolecular component attached to thymidylate synthase in methotrexate-resistant S. faecium. 4) Determine the inhibitory activity of polyglutamate derivatives of 5,8-dideaza-10-propargyl folate for thymidylate synthase. 5) Determine the antimicrobial and enzyme inhibitory potency of diastereoisomers of 10-methyl-10-deazaminopterin. 6) To prepare S. faecium thymidylate synthase in a form suitable for X-ray studies. The methodologies to be employed include: microbiological assay, enzyme assay, affinity chromatography, high performance liquid chromatography, sucrose gradient sedimentation, and RNA sequencing. Through collaborative arrangements we have access to X-ray crystallography, large scale fermentations, organic synthesis and experimental chemotherapy in mouse tumor systems. Health relatedness- Thymidylate synthase is inhibited by a metabolite of the antitumor agent fluorouracil and dihydrofolate reductase is inhibited by methotrexate (antitumor agent) and trimethoprim (antibacterial agent). Our work leads to information which is useful in guiding the efforts of organic chemists to synthesize improved compounds.